Method to predict heritable canine non-contact cruciate ligament rupture

ABSTRACT

Method and kits for diagnosing propensity to non-contact cranial cruciate ligament rupture (CCLR) in a dog are described. The method includes isolating genomic DNA from a dog and then analyzing the genomic DNA from step for a single nucleotide polymorphism occurring in selected loci that have been determined to be associated with the CCLR phenotype via a genome-wide association study.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of co-pending application Ser. No. 15/010,491,filed Jan. 29, 2016, now U.S. Pat. No. 10,131,950, issued Nov. 20, 2018,which claims priority to provisional application Ser. No. 62/109,336,filed Jan. 29, 2015, which is incorporated herein by reference.

BACKGROUND

The domestication of the dog and subsequent development of many dogbreeds has been declared one of the greatest genetic experiments everconducted by human beings. [Ostrander, E. A., Wayne, R. K. (2005).] Thecanine genome. Genome Research. 15: 17061716. There are now over 300unique breeds of dog. These breeds have been purposefully developed withspecific behavioral and physical traits in mind, thereby showcasing theincredible genetic diversity of the species—from Great Danes toChihuahuas. However, an unintended consequence of breed development isan increased incidence of disease states within certain breed. Many ofthe same disease states seen in certain dog breeds are also seen inhuman beings. Notably, the reduced genetic diversity in purebred dogshas generated stretches of linkage disequilibrium (LD) that are 40 to100 times longer in dogs than in humans. [Karlsson, E. K., Lindblad-Toh,K. (2008). Leader of the pack: gene mapping in dogs and other modelorganisms. Nature Reviews Genetics. 9: 713-725.] This presents a uniqueopportunity to study the genetic predisposition to disease moreefficiently by first identifying associations in dogs and using thatknowledge to inform human medical research.

Cruciate ligament rupture is one condition that occurs frequently inboth dogs and humans. The cranial cruciate ligament (CCL) is one of twointra-articular ligaments in the canine stifle (knee) joint, the otherbeing the caudal cruciate ligament. The CCL is analogous to the humananterior cruciate ligament (ACL), both anatomically and functionally.Both canine populations and human populations experience a conditionwhere the CCL/ACL ruptures without a traumatic force. This is known asnon-contact cruciate ligament rupture. See Alentorn-Geli, E., Myer, G.D., Silvers, H. J., Samitier, G., Romero, D., Lazaro-Haro, C., Cugat, R.(2009). Prevention of non-contact anterioro curicate ligament injuriesin soccer players. Part 1: Mechanisms of injury and underlying riskfactors. Knee Surgery, Sports, Traumatology, Arthroscopy. 17:705-729.Canine non-contact cranial cruciate ligament rupture (CCLR) is the mostcommon cause of pelvic limb lameness in dogs. CCLR is diagnosed inapproximately 20% of canine cases seen for lameness at universityinstitutions. [Wilke, V. L., Robinson, D. A., Evans, R. B., Rothschild,M. F., Conzemius, M. G. (2005). Estimate of the annual economic impactof treatment of cranial cruciate ligament in jury in dogs in the UnitedStates. Journal of the American Veterinary Medical Association. 227(10):1604-7.]The canine condition is characterized by progressive stiflejoint synovitis and osteoarthritis that leads to gradual fraying andeventual mid-substance rupture of the cranial cruciate ligament.Instability of the stifle joint as a result of CCLR is oftendebilitating and requires surgical treatment. The cost of surgery andpain management has a large economic impact. It has been estimated thatAmerican pet owners alone spend more than $1 billion per year on CCLRmanagement [Wilkie et al., supra]. When a dog presents with one stableand one unstable stifle, evidence of disease can often be found in thestable joint. [Bleedorn, J. A., Greuel, E. N., Manley, P. A, Schaefer,S. L., Markel, M. D., Holzman, G., Muir, P. (2011). Synovitis in dogswith stable stifle joints and incipient cranial cruciate ligamentrupture: A cross-sectional study. Veterinary Surgery. 40: 531-543.] Morethan 50% of dogs with unilateral CCLR will ultimately go on to rupturethe contra-lateral ligament. [Muir, P., Schwartz, Z., Malek, S.,Kreines, A., Cabrera, S. Y., Buote, N. J., Bleedorn, J. A., Schaefer, S.L., Holzman, G., Hao, Z. (2011) Contralateral cruciate survival in dogswith unilateral non-contact cranial cruciate ligament rupture. PLoS ONE.6(10): e25331.] While surgical stabilization does lead to clinicalimprovement, it does not cure the underlying mechanism that led toligament degeneration. Thus even with surgical interventionosteoarthritis will continue to develop in the joint over time.[Girling, S. L., Bell, S. C., Whitelock, R. G., Rayward, R. M., Thomson,D. G., Carter, S. C., Vaughan-Thomas, A., Innes, J. F. (2006). Use ofbiochemical markers of osteoarthritis to investigate the potentialdisease-modifying effect of tibial plateau levelling osteotomy. Journalof Small Animal Practice. 47: 708-714.]

While several hypotheses have been investigated, the mechanismunderlying the cruciate rupture condition in dogs and humans remainsunclear. Risk factors for disease initiation and disease progression indogs have been investigated. Neutering, weight, and gender have all beeninvestigated as risk factors for disease initiation. However, the mostimportant risk factor for disease initiation in dogs is breed. Theprevalence of CCLR in the Newfoundland, Labrador Retriever, and Boxerhas been estimated at 8.9%, 5.79%, and 5.24% respectively. In contrast,other breeds, such as the Greyhound and Old English Sheepdog, experiencemuch lower prevalence of CCLR (0.5% and 0.97%, respectively). TheLabrador Retriever breed has greater stifle joint laxity and a weakerCCL as compared to the Greyhound. Family-based pedigree studies indicatethat heritability of CCLR is high for a complex trait. Data reveal aheritability estimate of 0.27 in the Newfoundland and 0.28 in the Boxer.Human medical research has also begun to look into genetics as apotential risk factor for ACL rupture. Individuals with a blood relativewho has ruptured their ACL are at two-times (2×) greater risk ofrupturing their own. Recent research in humans suggests that a rareCOL1A1 gene variant may be protective against ACL rupture in youngathletes. See Clements, D. N., Kennedy, L. J., Short, A. D. Barnes, A.,Ferguson, J., Ollier, W. E. R. (2011). Risk of canine cranial curicateligament rupture is not associated with the major histocompatibitiltycomplex. Veterinary and Comparative Orthopaedics and Traumatology. 1-3;Hayashi, Kei., Manley, P. A., Muir, P. (2004). Cranial cruciate ligamentpathophysiology in dogs with cruciate disease: A review. Journal of theAmerican Animal Hospital Association. 40: 385-390; Witsberger, T.,Villamil, J., Schultz, L., Hahn, A., Cook, J. (2008). Prevalence of andrisk factors for hip dysplasia and cranial cruciate ligament deficiencyin dogs. Journal of the American Veterinary Medicial Association. 232(12): 1818-1824; Whitehair, J. G., Vasseur, P. B., Willits, N. H.(1993). Epidemiology of cranial cruciate ligament rupture in dogs.Journal of the American Veterinary Medical Association. 203: 1016-1019;Wilke, V. L., Conzemius, M. G., Kinghorn, B. P., Macrossan, P. E., Cai,W., Rothschild, M. F. (2006). Inheritance of rupture of cranial cruciateligament in Newfoundlands. Journal of the American Veterinary MedicalAssociation. 228: 61-64; Nielen, A. L., Janss, L. L., Knol, B. W.(2001). Heritability estimations for diseases, coat color, body weight,and height in a birth cohort of Boxers. American Journal of VeterinaryResearch. 62,8: 1198-1206; Flynn, R. K., Pedersen, C. L., Birmingham, T.B., Kirkley, A., Jackowski, D., Fowler, P. J. (2005). The familialpredisposition toward tearing the anterior cruciate ligament. TheAmerican Journal of Sports Medicine.33: 23-28; Posthumus, M., September,A. V., Keegan, M., O'Cuinneagain, D. , Van der Merwe, W., Schwellnus, M.P., Collins, M. (2009) Genetic risk factors for anterior cruciateligament ruptures: COL1A1 gene variant. British Journal of SportsMedicine. 43: 352-356; and Khoschnau, S., Melhus, H., Jacobson, A.,Rahme, H., Bengtsson, H., Ribom, E., Grundberg, E., Mallmin, H.,Michaelsson, K. (2008). Type I collagen alpha1 sp1 polymorphism and therisk of cruciate ligament ruptures or shoulder dislocations. TheAmerican Journal of Sports Medicine. 36: 2432-2436.

Two studies have mapped the CCLR trait to the canine genome.Associations with CCLR were reported on canine chromosomes 3, 5, and 15using a broad genomic scan of 495 microsatellite markers in Newfoundlanddogs. [Wilke, V. L., Zhang, S., Evans, R. B., Conzemius, M. G.,Rothschild, M. F. (2009). Identification of chromosomal regionsassociated with cranial cruciate ligament rupture in a population ofNewfoundlands. American Journal of Veterinary Research. Vol. 70,8:1013-1017.] More recently, a high-resolution genome-wide associationstudy (GWAS) for CCLR, also in the Newfoundland breed, found singlenucleotide polymorphism (SNP) associations on canine chromosomes 1, 3,10, 12, 22, and 33. [Baird, A. E. G., Carter, S. D., Innes, J. F.,Ollier, W., Short, A. (2014). Genome-wide association study identifiesgenomic regions of association for cruciate ligament rupture inNewfoundland dogs. Animal Genetics. 45, 4: 542-549.] The 65 mostsignificant SNPs were re-genotyped with a custom chip array, whichidentified significant regions on chromosomes 1, 3, and 33. Theseregions contained several genes that are highly expressed in the nervoussystem, suggesting a potential neuronal signaling component to CCLRrisk. The Baird et al. GWAS was unable to replicate results from theearlier Wilkie et al. microsatellite marker study.

SUMMARY OF THE INVENTION

To advance understanding of the genetic risk factors contributing toCCLR, a GWAS was performed to identify candidate genomic regionsassociated with the CCLR trait. To take advantage of the long-range LDpresent in dogs, the GWAS was limited to a single high-risk breed, theLabrador retriever, which has a high prevalence of CCLR. The Labradorretriever is also the most common breed in the United States accordingto records of the American Kennel Club.

As disclosed herein, CCLR is associated with multiple regions of thecanine genome. Thus, by analyzing dogs for mutations in theseCCLR-associated regions, the propensity of their progeny to carry thetrait, and thus to experience CCLR, can be determined. This informationcan then be used to guide breeding efforts to reduce the occurrence ofCCLR.

Thus, disclosed herein is a method for diagnosing propensity tonon-contact cranial cruciate ligament rupture (CCLR) in a dog. Themethod comprises isolating genomic DNA from a dog and then analyzing thegenomic DNA for single nucleotide polymorphisms occurring within, or ina genomic interval of about 2 Mb upstream or downstream of, at least onelocus revealed herein to be associated with the CCLR phenotype. Theseloci include BICF2P1126668, BICF2P260555, BICF2P599385, BICF2P1465216,BICF2S23135243, BICF2P170661, TIGRP2P78405, BICF2P890246, BICF2P401973,BICF2G630114782, BICF2G630815470, BICF2G630815474, BICF2S23448539,BICF2P1121006, BICF2G630371956, BICF2S2356299, BICF2P526639,BICF2P154295, BICF2P412007, BICF2S23645462, BICF2G630373050, andBICF2P471347. The dog has an increased propensity for CCLR when five ormore SNPs (or 10 or more, or 15 or more, or 20 or more) are detected inthe dog's genomic DNA.

Also disclosed herein is a method for diagnosing propensity tonon-contact cranial cruciate ligament rupture (CCLR) in a dog. Here, themethod focuses on mutations in specific genes. The method comprisesisolating genomic DNA from a dog. The DNA is then analyzed for singlenucleotide polymorphisms occurring within, or in a genomic interval ofabout 2 Mb upstream or downstream of, at least one gene selected fromthe group consisting of CDH18, DPPA3, UBE2D1, ASS1, SPRED2, DLC1, DYSF,SCGB2, CHST2, SLC15A, ANO2, ERRFI1, SCGB2, and TRIM42. Again, the doghas an increased propensity for CCLR when five or more SNPs (or 10 ormore, or 15 or more, or 20 or more) are detected in the dog's genomicDNA.

Also disclosed herein are kits for diagnosing the propensity tonon-contact cranial cruciate ligament rupture (CCLR) in a dog. The kitscomprise oligonucleotide probes or primers dimensioned and configured tobind selectively to single nucleotide polymorphism occurring within, orin a genomic interval of about 2 Mb upstream or downstream of at leastone locus selected from the group consisting of BICF2P1126668,BICF2P260555, BICF2P599385, BICF2P1465216, BICF2S23135243, BICF2P170661,TIGRP2P78405, BICF2P890246, BICF2P401973, BICF2G630114782,BICF2G630815470, BICF2G630815474, BICF2S23448539, BICF2P1121006,BICF2G630371956, BICF2S2356299, BICF2P526639, BICF2P154295,BICF2P412007, BICF2S23645462, BICF2G630373050, and BICF2P471347.Instructions for use of the kit are typically included.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 1to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All references to singular characteristics or limitations of the presentinvention shall include the corresponding plural characteristic orlimitation, and vice-versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.Unless otherwise stated, the indefinite articles “a” and “an” mean “oneor more.” When referring to a previously stated element, the definitearticle “the” does not limit the stated definition of “a” and “an,” asbeing “one or more.”

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The methods and kits disclosed herein can comprise, consist of, orconsist essentially of the essential elements and limitations describedherein, as well as any additional or optional steps, ingredients,components, or limitations described herein or otherwise useful ingathering, preparing, and sequencing genomic DNA for analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that the Labrador Retriever is a dog breedwith a relatively low amount of inbreeding. Whiskers represent theminimum and maximum values for each analysis method (n=237 dogs).

FIG. 2 is a series of graphs demonstrating that linear mixed model GWAScorrects for population structure and identifies 98 ACL associated lociexplaining a large proportion of phenotypic variance. For each linearmixed model (LMM), the QQ plots show no evidence of populationstratification relative to the expected distribution. Permutationtesting with each model determined genome-wide significance at panel (a)P<3.63E-7 for GCTA (Genome-wide Complex Trait Analysis), λ=0.987; panel(b) P<6.097E-7 for GEMMA (Genome-wide Efficient Mixed ModelAssociation), λ=0.994; and panel (c) P<4.01E-7 for PUMA (PenalizedUnified Multiple-locus Association, λ=1.012. The plots representanalysis of 118,992 SNPs from 98 cases and 139 phenotype-negativecontrols. Panel (d): with GCTA, 36 loci have P<5E-4, with the mostsignificant locus located in CFA 26, which did not meet genome-widesignificance defined by minimum p-values from permutation testing. Panel(e): with GEMMA, 47 loci have P<5E-4, with the locus on CFA 26 meetinggenome-wide significance defined by minimum p-values from permutationtesting. Panel (f): with PUMA, 65 loci were significant at P<5E-4 andthe locus on CFA 26 exceeded genome-wide significance defined by minimump-values from permutation testing.

FIG. 3 is a graph showing that phenotype variance was explained to alarge degree by the associate genomic loci. Loci identified by linearmixed model (LMM) analysis were broadly defined as SNPs with r²>0.5within 5 Mb of the peak SNP. For GCTA, 36 loci in 72.7 Mb of the genomeexplained 48.09% of the phenotypic variance. For GEMMA, 47 loci in 82.7Mb of the genome explained 55.88% of the phenotypic variance. For PUMA,65 loci in 86.58 Mb of the genome explained 50.28% of the phenotypicvariance in the ACL rupture trait.

FIG. 4 and FIG. 5 show that genetic risk scoring using GWAS associatedloci from linear mixed model analysis with GEMMA predicts ACL rupturedisease risk in both case and control Labrador Retriever dogs. FIG. 4 isa graph showing the distribution of the number of ACL rupture risk lociin case and control groups of Labrador Retriever dogs. The number ofrisk alleles in cases and controls is significantly different(P<2.2E-16). FIG. 5 is a graph depicting ACL rupture odds ratios ofweighted genetic risk scores (wGRS) relative to the first quartile.Vertical bars represent the 95% confidence intervals. * Odds ratio issignificantly different from the reference first quartile.

DETAILED DESCRIPTION Abbreviations and Definitions:

CCLR: cranial cruciate ligament rupture (non-contact).

EDTA: Ethylenediaminetetraacetic acid.

GEMMA: Genome-wide efficient mixed model association.

GenABEL is an online project is to provide a free framework forcollaborative, robust, transparent, and open source-based development ofstatistical genomics methodology. See http://www.genabel.org/.

GRAMMAR-Gamma is a genomic analysis program which is available throughGenABEL. See also Svishcheva, G. R., Axenovich, T. I., Belonogova, N.M., van Duijn, C. M., and Aulchenko, Y. S. (2012) “Rapid variancecomponents—based method for whole-genome association analysis,” NatureGenetics 44:1166-1170.

GWAS: Genome-wide association study. A genome-wide association study isan analysis of genetic variation at specified loci in differentindividuals to see if any variant(s) is (are) associated with aphenotypic trait. As the name indicates, genetic markers across thecomplete genome of each individual test subject are tested to findgenetic variations associated with a particular disease, in this caseCCLR in dogs. Once new genetic associations are identified, theinformation is used to detect, treat and/or prevent the disease. Suchstudies are particularly useful in finding genetic variations thatcontribute to common, but complex diseases.

LD: Linkage disequilibrium. Linkage disequilibrium is the non-randomassociation of alleles at two or more loci that descend from single,ancestral chromosomes.

MDS: multidimensional scaling.

MLM, LLM (synonymous): mixed linear model, linear mixed model,respectively.

P3D: Population parameters previously determined.

PLINK: PLINK is a free, open-source whole genome association analysisprogram that performs a range of large-scale genomic analyses in acomputationally efficient manner. The PLINK software was developed (andcontinues to be refined) by Shaun Purcell and others at the Center forHuman Genetic Research, Massachusetts General Hospital, and the BroadInstitute of Harvard & MIT. PLINK v.1.9 is available online as of May15, 2014 at http://pngu.mgh.harvard.edu/˜purcell/plink/.

SNP: Single nucleotide polymorphism.

TASSEL: Trait analysis by association, evolution and linkage.

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in genetics, genomics,and molecular biology may be found in Benjamin Lewin, “Genes V,”published by Oxford University Press, 1994 (ISBN 0-19-854287-9) andKendrew et al. (eds.), “The Encyclopedia of Molecular Biology,”published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9).

Canine Samples and Phenotyping:

DNA was isolated from client-owned Labrador Retrievers using blood orbuccal swabs. A four-generation pedigree was collected from each dog toensure purebred status and identify siblings, which were excluded fromthe GWAS. Each dog underwent an orthopaedic examination that includedassessment of knee stability [Muir P. Physical examination of lame dogs.Comp Cont Ed Pract. Vet 1997; 19: 1149-1161]. Radiographs of theaffected knee(s) were also assessed in cases. In addition, lateralweight-bearing knee radiographs [Kim S E, Lewis D D, Pozzi A, Seibert RL, Winter M D. Radiographic quantitative assessment of cranial tibialsubluxation before and after tibial plateau leveling osteotomy in dogs.Am J Vet Res. 2011; 72: 420-416] were made to screen phenotype-negativecontrol dogs. While it is not possible to identify the cruciateligaments radiographically in the dog, compression of the infrapatellarfat pad in the knee by synovial effusion and knee osteophytosis aredegenerative changes typically associated with ACL rupture [Chuang C,Ramaker M A, Kaur S, Csomos R A, Kroner K T, Bleedorn J A, et al.Radiographic risk factors for contralateral rupture in dogs withunilateral cranial cruciate ligament rupture]. Dogs were consideredcases if anterior translation of the tibia was detected clinically andradiographic signs were consistent with ACL rupture. Labrador Retrievers≥8 years of age have less than a 6% chance of developing ACL rupture[Reif U, Probst C W. Comparison of tibial plateau angles in normal andcranial cruciate deficient stifles of Labrador retrievers. Vet Surg.2003; 32: 385-389]. Therefore, control dogs were ≥8 years of age with anormal orthopaedic clinical exam and normal knee radiographs. Habitualactivity of each dog was documented using a questionnaire.

Genome-Wide Association:

Genome-wide SNP genotyping was performed in 98 cases and 139 controlsusing the 11lumina CanineHD BeadChip, which genotypes 173,662 SNPsevenly spaced across the genome. Data underwent quality controlfiltering using PLINK [Chang C C, Chow C C, Tellier L C A M, VattikutiS, Purcell S M, Lee J J. Second-generation PLINK: rising to thechallenge of larger and richer datasets. GigaScience. 2015; 4:7]. Allsamples had a genotyping call rate of ≥95%. 49,859 SNPs were excludedbecause minor allele frequency (MAF) was ≤0.05 and 7,468 SNPs wereexcluded because of a low genotyping rate (≤95%). 153 SNPs were excludedbecause of deviation from Hardy-Weinberg equilibrium at P<1E-07. 118,992SNPs were used for further analysis.

To account for ancestral population structure and family relatedness inthe study dogs, single marker linear mixed model (LMM) analysis wasperformed using GCTA (Genome-wide Complex Trait Analysis) [Yang K, Lee SH, Goddard M E, Visscher. GCTA: A tool for genome-wide complex traitanalysis. Am J Hum Genet. 2011; 88: 76-82] and GEMMA (Genome-wideEfficient Mixed Model Association) [Zhou X, Stephens M. Genome-wideefficient mixed-model analysis for association studies. Nat Genet. 2012;44: 821-824], software tools optimized for complex trait GWAS. PenalizedUnified Multiple-locus Association (PUMA), in which all SNPs areanalyzed together, was also used to aid detection of weaker associationsoften found in complex traits [Hoffman G E, Logsdon B A, Mezey J G.PUMA: A unified framework for penalized multiple regression analysis ofGWAS data. PLoS Comput Biol. 2013; 9: e1003101]. We used logisticregression and a 2D-MCP penalty for this analysis [Hoffman G E, LogsdonB A, Mezey J G. PUMA: A unified framework for penalized multipleregression analysis of GWAS data. PLoS Comput Biol. 2013; 9: e1003101;Zhang C H. Nearly unbiased variable selection under minimax concavepenalty. Ann Stat. 2010; 38: 894-942]. In the PUMA analysis, the first20 eigenvectors were used as covariates in the association analysis tocorrect for population structure. Eigenvectors were obtained byprincipal component analysis using GCTA. Because neutering has asignificant effect on risk of ACL rupture, it was included as acovariate with the GEMMA, GCTA, and PUMA analyses.

Genome-Wide Significance:

We defined genome-wide significance using permutation testing. Use of aBonferroni correction for the number of SNPs tested is too conservativein dog breeds, as extensive LD means that SNPs are often inherited inhaplotype blocks [Lindblad-Toh K, Wade C M, Mikkelsen T S, Karlsson E K,Jaffe D B, Kamal M, et al. Genome sequence, comparative analysis, andhaplotype structure of the domestic dog. Nature. 2005; 438: 803-819]. Wedefined genome-wide significance by randomly permuting the phenotypesand re-running the GWAS LMM 1,000 times. Genome-wide significance wasdefined by identifying the 5% quantile of the set of minimum P-valuesfrom the GWAS permutations. Additionally, we calculated the number ofhaplotype blocks in the Labrador Retriever SNP data using PLINK, usingLD windows of 500 kb, 1 Mb, and 5 Mb and used the number of haplotypeblocks to estimate genome-wide significance by Bonferroni correction ofP<0.05. To facilitate further dissection of genetic variants associatedwith the ACL phenotype, we also identified a larger set of candidate ACLrupture regions at P<5E-04 [Karlsson E K, Sigurdsson S, Ivansson E,Thomas R, Elvers I, Wright J, et al. Genome-wide analyses implicate 33loci in heritable dog osteosarcoma, including regulatory variants nearCDKN2A/B. Genome Biol. 2013; 14: R132]. Although some of the regionsincluded may not be true associations, this would likely weaken ratherthan strengthen the gene set and pathway analyses, leading to falsenegatives rather than false positives.

Defining Associated Loci in the Genome:

Linkage-disequilibrium (LD) clumping using PLINK was used to defineregions of association with the ACL rupture trait from the GWAS results.LD clumping defined regions around SNPs associated at P<5E-04. Regionswithin 1 Mb of the index SNP (r²>0.8 and P<0.01). We also used GCTA toexplain the phenotype variance explained by the associated loci, whichwere defined as SNPs with r²>0.2 within 5 Mb of the peak SNP in eachlocus [Tang R, Noh H J, Wang D, Sigurdsson S, Swofford R, Perlosko M, etal. Candidate genes and functional noncoding variants identified in acanine model of obsessive-compulsive disorder. Genome Biol. 2014; 15:R25].

For complex trait GWAS with a large number of risk loci, loci that arenot discovered are expected to have smaller effect sizes in a secondgeneration GWAS, because those with larger effect sizes will have beenidentified in the first round of GWAS. To estimate the number of riskloci that are likely associated with ACL rupture, we used INPower. Oddsratios were corrected for the winner's curse before INPower analysis wasperformed. See Park J-H M, Wacholder S, Gail M, Peters U, Jacobs K B,Chanock S J, et al. Estimation of effect size distribution fromgenome-wide association studies and implications for future discoveries.Nat Genet. 2010; 42: 570-575 and Ghosh A, Zou F, Wright F A. Estimatingodds ratios in genome scans: An approximate conditional likelihoodapproach. Am J Human Genet. 2008; 82: 1064-1074.

Genetic Risk Score Computation:

Two approaches were used to calculate the genetic risk scores (GRS), asimple risk alleles count method (cGRS) and a weighted method (wGRS)[Chen H, Poon A, Yeung C, Helms C, Pons J, Bowcock A M, et al. A geneticrisk score combining ten psoriasis risk loci improves diseaseprediction. PLoS One. 2011; 6: e19454]. The wGRS weights each riskallele by the logarithm odds ratio (Log(OR)) for that allele. The wGRSis a linear combination of the number of risk alleles weighted by theLog(OR) as coefficients. The Mann-Whitney U test was used to comparecGRS scores for each LMM in case and control groups. To estimate thetotal risk captured by the genetic risk scoring for each LMM, wecalculated the odds ratios according to the wGRS quartiles. We alsomeasured the discriminative power attributable to the GRS by plottingreceiver operating characteristic (ROC) curves and calculated the areaunder the curve (AUC) for the Labrador Retriever case and control dogs.AUC 95% confidence intervals were calculated using 2000 stratifiedbootstrap replicates. An R software package (http://www.r-project.org/)was used for these analyses.

Pathway Analysis:

Pathway analysis was performed with two methods. DAVID [Huang D W,Sherman B T, Lempicki R A. Systematic and integrative analysis of largegene lists using DAVID bioinformatics resources. Nat Protoc. 2009; 4:44-57] analyses were run on the ACL rupture loci identified from ourGWAS. ACL rupture loci were transposed to CanFam 3.1 coordinates(genome.ucsc.edu/cgi-bin/hgLiftOver) with 500 kB flanks added to thestart and end and gene size correction turned on [Tang R, Noh H J, WangD, Sigurdsson S, Swofford R, Perlosko M, et al. Candidate genes andfunctional noncoding variants identified in a canine model ofobsessive-compulsive disorder. Genome Biol. 2014; 15: R25]. A list ofgenes from the liftover coordinates was then analyzed. Probabilityvalues were evaluated after Benjamini correction with DAVID.

Pathway analysis with INRICH was performed on canFam2 intervals using amap file lifted over from the canFam3.1 Broad Improved Canine Annotationcatalog (UCSC Genome Browser) [Lee P H, Dushlaine C O, Thomas B, PurcellS M. INRICH: interval-based enrichment analysis for genome-wideassociation studies. Bioinformatics. 2012; 28: 1797-1799]. We used1,000,000 permutations matched for region size, SNP density, and genenumber. INRICH reports significance for each gene set and theexperiment-wide significance, correcting for the number of gene sets(P_(corr)). We considered P_(corr)<0.05 to be significant. We testedgene sets from the KEGG (Kyoto Encyclopedia of Genes and Genomes), GeneOntology, and MSigDB (Molecular Signatures Database).

Heritability Estimation:

Narrow sense heritability was estimated from SNPs using the BGLRstatistical package [Pérez P, de los Campos G. Genome-wide regressionand prediction with the BGLR statistical package. Genetics. 2014; 198:483-495]. SNPs with missing genotypes were filtered out using PLINK.Heritability estimation was performed using SNPs. A genomic best linearunbiased prediction (GBLUP) model was fitted using a SNP-derived genomicrelationship matrix using a non-parametric reproducing kernel Hilbertspaces (RKHS) method as described in Perez (2014). Broad senseheritability was also estimated using a data matrix prepared frompedigrees. To fit the model, 30,000 iterations of the Gibbs sampler wereused with burn-in of 5,000 iterations. A correction factor was used totransform the heritability estimate on the observed scale from theregression model to the liability scale for a binary trait [Zhou X,Caronetto P, Stephens M. Polygenic modeling with Bayesian sparse linearmixed models. PLoS Genetics. 2013; 9: e1003264] and a populationprevalence of 0.0579 [Witsberger T H, Villamil J A, Schultz L G, Hahn AW, Cook J L. Prevalence of and risk factors for hip dysplasia andcranial cruciate ligament deficiency in dogs. J Am Vet Med Assoc. 2008;232: 1818-1824] was used for this correction.

Linkage Disequilibrium Analysis:

After obtaining the results from each MLM, LD-based clumping wascalculated in PLINK to define associated regions in LD with the mostsignificant SNPs (r²>0.5, within 2 Mb of the associated SNP). Thesesettings were modified from another GWAS for a complex trait in dogs.[Karlsson et al. (2013). Genome-wide analyses implicate 33 loci inheritable dog osteosarcoma, including regulatory variants near CDKN2A/B.Genome Biology. 14:R132.] These regions were then investigated with theNCBI Canine Genome Map Viewer to identify nearby genes using the CanFam3.0 reference sequence.

GWAS Population of Labrador Retrievers:

We genotyped 237 Labrador Retrievers using the Illumina CanineHDBeadChip, removing SNPs with call rates of <95%. No dogs were removedafter SNP filtering. The final dataset contained 118,992 SNPs from 98cases and 139 phenotype-negative controls. Median inbreeding coefficientwas 0.025 (FIG. 1). The ratio of females to males in the case andcontrol groups was 0.92 and 0.83 respectively. Of the 114 females, 99were ovariohysterectomized (0.87). Of the 123 males, 96 were castrated(0.78). Mean age of the dogs in the case and control groups was 6.0±2.5years and 10.4±1.7 years, respectively.

GWAS Identifies 98 Regions Associated with Anterior Cruciate LigamentRupture:

We tested for association between ACL rupture and SNPs with a MAF>0.05in the Labrador Retriever breed, controlling for cryptic relatedness andpopulation structure using LMM analysis with three programs, including apenalized multiple regression method for improved detection of weakassociations. We identified all SNPs with either significant associationbased on analysis of 1,000 random phenotype permutations to definegenome-wide significance (P<1.549E-06 for GCTA, P<6.097E-07 for GEMMAand P<4.35E-07 for PUMA) or suggestive association (P<5.00E-04; FIG. 2)and defined regions of associated using linkage disequilibrium. SeeTables 1 and 2. Control dogs were considered phenotype-negative becauseof the selection criteria used for recruitment. We identified 21,713;21,754; and 21,861 haplotype blocks in the Labrador Retriever genomewith LD windows of 5 Mb, 1 Mb, and 5 kb respectively, yielding agenome-wide significance estimate of P<2.29E-06 to P<2.30E-06.

TABLE 1 Anterior cruciate ligament rupture associated loci identified byGWAS in the Labrador Retriever, a dog breed with a high diseaseprevalence Risk Size SNP chr Position P LMM allele f(A) f(U) OR Regionstart-end (kB) Genes BICF2G630500368 24 30241088 2.76E−07 1, 2, 3 G 0.830.66 2.56 30241088-30245795 5 BPI, LBP, RALGAPB, ADIG, ARHGAP40,SLC32A1, ACTR5, PP1R16B, FAM83D, DHX35 BICF2P1121006 18 542795781.11E−05 1, 2, 3 A 0.63 0.42 2.28 No LD Many (30 genes) BICF2S2356299 2730557856 2.21E−05 2, 3 A 0.43 0.27 2.03 No LD None BICF2P483191 2921601273 2.31E−05 1, 2, 3 C 0.73 0.51 2.54 No LD SULF1 SLCO5A1, PRDM14,NCOA2, TRAM1 BICF2P50610 11 32270617 2.75E−05 3 A 0.29 0.19 1.731939564-32270617 331 C11H9orf123, PTPRD BICF2P890246 9 534279073.23E−05 1, 2 A 0.16 0.36 2.99 53427907-53432248 4 Many (26 genes)BICF2S23324965 6 14077648 3.36E−05 3 G 0.68 0.60 1.42 14077648-1409205714 NPTX2, BAIAP2L1, BRI3, TECPR1, BHLHA15, LMTK2, CCZ1, RSH10B, PMS2,AIMP2, ANKRD61, EIF2AK1, USP42, CYTH3 BICF2P544126 24 29772193 4.09E−053 G 0.94 0.87 2.28 29772193-29794411 22 CTNNBL1, VSTM2L, TTI1, RPRD1B,TGM2, KIAA1755 BICF2P526639 27 39217437 4.12E−05 2, 3 G 0.23 0.12 2.1839211186-39217437 6 A2M BICF2P1462185 20 15053718 4.89E−05 3 A 0.85 0.741.90 14838270-15053718 215 EDEM1, ARL8B BICF2P1208798 9 126712175.49E−05 1, 2 G 0.56 0.36 2.27 No LD EFCAB13, ITGB3, MYL4, CDC27,KANSL1, MAPT, SPPL2C, CRHR1, NSF, WNT3 BICF2G630175389 4 842609065.87E−05 1, 2 A 0.83 0.68 2.28 No LD CDH10 BICF2S24415473 3 869740427.07E−05 1, 2 G 0.40 0.26 1.97 86948527-86974042 26 STIM2, PGM2, RELL1,C3H4orf19, NWD2 BICF2G630412697 30 3126573 7.22E−05 1, 3 G 0.96 0.864.23 No LD COR4F22P, COR4F25, COR4F24P, COR4T2P BICF2P498515 6 758485377.89E−05 1, 2, 3 A 0.16 0.06 3.11 No LD LRRIQ3 BICF2P792911 26 228949618.55−05 1, 2, 3 G 0.44 0.27 2.14 No LD TPST2, CRYBB1, CRYBB4BICF2G630810143 6 11130832 9.46E−05 3 A 0.44 0.32 1.72 11130832-1117714946 DTX2, UPK3B, UPK3BL, RASA4, LRWD1, ALKBH4, ORAI2, PRKRIP1, SH2B2,CUX1, MYL10, COL26A1, RABL5, RIS1, CLDN15, ZNHIT1, PLOD3 BICF2P564273 355250188 1.07E−04 1, 2, 3 A 0.70 0.52 2.16 No LD ACAN, HAPLN3, MFGE8,ABHD2, RLBP1, FANCI, POLG, TRNAR-UCG, RHCG, TICRR, KIF7, PLIN1, PEX11A,WDR93, MESP1, MESP2, ANPEP, AP3S2, ARPIN TIGRP2P297337 22 582014521.08E−04 1, 2, 3 A 0.44 0.27 2.2 No LD EFNB2, ARGLU1 BICF2G630658881 217582214 1.09E−04 1, 2, 3 G 0.49 0.32 2.12 7581714-8383209 JRKL, CCDC82,MAML2, MTMR2, CEP57, FAM76B, SESN3 Note: OR odds ratio calculated fromPLINK. LMM Linear mixed model 1—GCTA, 2—GEMMA, 3—PUMA. Data representthe twenty most significant loci of 98 associations with canine ACLrupture. SNP position and genomic regions are based on CanFam 2.0. Geneslists were derived from the SNP locus or LD block with 500kb flankingregions.

TABLE 2 Anterior cruciate ligament rupture associated SNPs identified byGWAS in the Labrador Retriever, a dog breed with a high diseaseprevalence Risk SNP chr Position P LMM Allele f(A) f(U) OR Regionstart-end Genes BICF2G630709791 1 10788643 1.64E−04 1 C 0.30 0.16 2.1810788643-11025688 RTTN, CD226, DOK6 BICF2S23147946 1 17290917 1.74E−04 1G 0.28 0.15 2.19 No LD BCL2, PHLPP1, ZCCHC2, TNFRSF11A, KIAA1468,PPIAP1, PIGN BICF2P181859 1 17840093 4.32E−04 1 A 0.21 0.09 2.60 No LDZCCHC2, TNFRSF11A, KIAA1468, PPIAP1, PIGN, CDH20 BICF2G630712921 119148000 4.94E−04 1 G 0.46 0.32 1.87 18645187-19148000 CDH20, MC4R,PMAIP1, CCBE1 BICF2G630713147 1 19274346 4.93E−04 1 A 0.37 0.24 1.8319253280-19274346 MC4R, PMAIP1, CCBE1 BICF2P818099 1 39021948 4.29E−04 3G 0.65 0.54 1.55 No LD SF3B5, STX11, TRNAL-UAA, UTRN BICF2S23638642 145229667 4.95E−04 2 G 0.18 0.07 3.07 No LD AKAP12, ZBTB2, RMND1, ARMT1,CCDC170, ESR1, SYNE1 BICF2P206910 1 46405864 3.25E−04 2 C 0.14 0.04 4.0546405864-46443183 MYCT1, VIP, BICF2S22959529 1 46443183 1.58E−04 1, 2 C0.14 0.03 4.78 FBX05, MTRF1L, RGS17 BICF2P1054044 2 20002181 2.28E−04 3G 0.85 0.77 1.74 No LD MTPAP, MAP3K8, LYZL1, TRNAK- CUU, BAMBI, WACBICF2S23533020 2 20501149 2.47E−04 3 G 0.92 0.84 2.33 20288541-20501149LYZL1, TRNAK- CUU, BAMBI, WAC, MPP7 BICF2P720951 2 62500174 4.61E−04 3 A0.26 0.16 1.82 62252040-62500174 GPR114, CCDC102A, DOK4, POLR2C, COQ9,CIAPIN1, CCL17, CX3CL1, CCL22, TRNAL-CAG, PLLP, ARL2BP, RSPRY1, FAM192A,CPNE2, NLRC5, HERPUD1, SLC12A3, NUP93, MT2A, MT-III, MT4, BBS2, OGFOD1,NUDT21, AMFR, GNAO1 TIGRP2P31530 2 80046637 1.65E−04 2, 3 G 0.67 0.551.69 80046637-80079758 C1QB, C1QC, BICF2P247448 2 80068322 1.48E−04 2, 3G 0.68 0.55 1.67 C1QA, EPHA8, BICF2P1066899 2 80079758 1.65E−04 2, 3 A0.67 0.55 1.69 ZBTB40, WNT4, CDC42, CELA3B, HSPG2, LDLRAD2, USP48,RAP1GAP, ALPL BICF2G630464857 2 87862734 2.00E−04 2 G 0.14 0.04 3.69 NoLD C2H1orf167, AGTRAP, DRAXIN, MAD2L2, FBXO6, FBXO44, FBXO2, PTCHD2,UBIAD1, MTOR, ANGPTL7, EXOSC10, SRM, MASP2, TARDBP, CASZ1, PEX14, DFFA,APITD1, PGD BICF2G630108404 3 39424250 4.96E−04 3 G 0.24 0.13 2.0639424250-39517119 NDN, MKRN3, BICF2S23148483 3 39469011 4.96E−04 3 G0.24 0.13 2.06 CHRNA7 BICF2P866702 3 39517119 4.96E−04 3 A 0.24 0.132.06 BICF2P1109077 3 48873558 4.26E−04 3 G 0.80 0.67 1.9048873558-48880579 MCTP2 BICF2P1431921 3 48878860 4.26E−04 3 G 0.80 0.671.90 BICF2P241884 3 48880579 4.26E−04 3 G 0.80 0.67 1.90 BICF2P564273 355250188 1.07E−04 1, 2, 3 A 0.70 0.52 2.16 No LD ACAN, HAPLN3, MFGE8,ABHD2, RLBP1, FANCI, POLG, TRNAR- UCG, RHCG, TICRR, KIF7, PLIN1, PEX11A,WDR93, MESP1, MESP2, ANPEP, AP3S2, ARPIN TIGRP2P46522 3 576989084.01E−04 3 C 0.70 0.54 1.91 57660110-57698908 PDE8A, RPS17, CPEB1,AP3B2, FSD2, WHAMM, HOMER2, FAM103A1, C3H5orf40, BTBD1, TM6F1, HDGFRP3,BNC1, SH3GL3 BICF2G630349775 3 77625147 4.26E−04 1, 2 A 0.18 0.07 2.90No LD TBC1D1, PGM2, RELL1, C3H4orf19, NWD2 BICF2S2342150 3 869485272.91E−04 1, 2 A 0.40 0.27 1.83 86948527-86974042 STIM2, TBC1D19,BICF2S24415473 3 86974042 7.07E−05 1, 2 G 0.40 0.26 1.97 CCKAR, SMIM20,SEL1L3 BICF2G630359517 3 91944314 3.77E−04 1, 2 C 0.51 0.36 1.82 No LDKCNIP4, PACRGL, SLIT2 BICF2G63058646 4 9120668 1.76E−04 1, 2, 3 G 0.130.04 3.71 No LD SLC35F3, KCNK1, KIAA1804, PCNXL2 BICF2P295392 4 145368704.61E−04 2 G 0.11 0.04 3.07 No LD BICC1, PHYHIPL, FAM13C BICF2G6301684734 74924050 2.60E−04 3 G 0.30 0.18 1.96 No LD NUP155, C4H5orf42, NIPBL,SLC1A3 BICF2G630175389 4 84260906 5.87E−05 1, 2 A 0.83 0.68 2.28 No LDCDH10 BICF2G630810143 6 11130832 9.46E−05 3 A 0.44 0.32 1.7211130832-11177149 DTX2, UPK3B, BICF2G630810159 6 11177149 9.46E−05 3 A0.44 0.32 1.72 UPK3BL, RASA4, LRWD1, ALKBH4, ORAI2, PRKRIP1, SH2B2,CUX1, MYL10, COL26A1, RABL5, FIS1, CLDN15, ZNHIT1, PLOD3 BICF2G6308101736 11181920 1.33E−04 3 G 0.61 0.51 1.50 11035074-11181920 SRCRB4D, ZP3,MOGAT3, NAT16 BICF2P170661 6 11439931 3.39E−04 3 A 0.31 0.22 1.57 No LDVGF, AP1S1, SERPINE1, TRIM56, ACHE, UFSP1, SRRT, TRIP6, SLC12A9, EPHB4BICF2P1354767 6 11462695 3.02E−04 3 G 0.48 0.38 1.53 11106977-11462695None BICF2P205255 6 11484772 4.75E−04 3 G 0.54 0.45 1.43 No LD ZANBICF2P1358119 6 13131171 3.74E−04 3 C 0.44 0.34 1.54 13131171-13182379AZGP1, GJC3, TRIM4, VN2R301P, CYP3A26, CYP3A4, OR0C09, ZSCAN25, FAM200A,ZKSCAN5, ZNF789, ZNF394, TRNAW- CCA, CPSF4, PTCD1, BUD31, PDAP1, ARPC1B,ARPC1A, MYH16, KPNA7, SMURF1, TRRAP, TMEM130 BICF2S23324965 6 140776483.36E−05 3 G 0.68 0.60 1.42 14077648-14092057 NPTX2, BAIAP2L1,BICF2S22961650 6 14092057 1.53E−04 3 G 0.68 0.61 1.35 BRI3, TECPR1,BHLHA15, LMTK2, CCZ1, RSPH10B, PMS2, AIMP2, ANKRD61, EIF2AK1, USP42,CYTH3 BICF2P498515 6 75848537 7.89E−05 1, 2, 3 A 0.16 0.06 3.11 No LDLRRIQ3 BICF2P1072682 7 53407178 4.09E−04 1, 2, 3 C 0.28 0.14 2.42 No LDNone BICF2P1090079 7 64389761 1.56E−04 1, 3 C 0.47 0.31 1.94 No LD CDH2,CHST9 BICF2P1208798 9 12671217 5.49E−05 1, 2 G 0.56 0.36 2.27 No LDEFCAB13, ITGB3, MYL4, CDC27, KANSL1, MAPT, SPPL2C, CRHR1, NSF, WNT3BICF2P890246 9 53427907 3.23E−05 1, 2 A 0.16 0.36 2.99 53427907-53432248KCNT1, SOHLH1, BICF2P139678 9 53432248 1.75E−04 1, 2 A 0.83 0.65 2.71LCN9, GLT6D1, LCN1, ABO, SURF6, MED22, RPL7A, SURF1, SURF2, SURF4,C9H9orf96, REXO4, ADAMTS13, CACFD1, SLC2A6, TMEM8C, ADAMTSL2, FAM163B,DBH, SARDH, VAV2, BRD3, WDR5, RXRA BICF2S23113199 10 46246942 3.57E−041, 3 A 0.63 0.46 1.95 No LD AFF3, REV1, EIF5B, TXNDC9, LYG1, LYG2,MRPL30, MITD1, TRNAK- CUU, LIPT1, TSGA10 BICF2P401973 10 653447723.79E−04 1 G 0.84 0.71 2.23 No LD FAM161A, CCT4, COMMD1, TRNAE- UUC,TMEM17, EHBP1 BICF2P454456 11 32175491 4.77E−04 3 A 0.19 0.13 1.6231831896-32175491 C11H9orf123, PTPRD BICF2P50610 11 32270617 2.75E−05 3A 0.29 0.19 1.70 31939564-32270617 C11H9orf123, PTPRD BICF2P531097 1132908558 3.58E−04 3 A 0.44 0.29 1.92 32908558-32922914 PTPRDBICF2P1290820 11 32922914 4.19E−04 3 G 0.44 0.30 1.91 BICF2P65003 1240691540 3.15E−04 2 G 0.71 0.61 1.61 40691540-41066621 SENP6, MYO6,IMPG1 BICF2G630606359 13 13352804 4.61E−04 2 G 0.69 0.57 1.6713352804-13503950 NUDCD1, ENY2, PKHD1L1, EBAG9, SYBU BICF2S23620879 1410265645 4.05E−04 3 C 0.5 0.37 1.71 9796003-10265645 COPG2, MEST, CEP41,CPA1, CPA5, CPA4, CPA2, SSMEM1, TMEM209, KLDHC10, TRNAM-CAU, ZC3HC1,UBE2H, NRF1, SMKR1, STRIP2, AHCYL2, SMO, TSPAN33, TNPO3, IRF5, KCP,ATP6V1F, FLNC BICF2G630519882 14 11686985 4.99E−04 3 A 0.49 0.36 1.7411675474-11686985 LRRC4, SND1, PAX4, FSCN3, ARF5, GCC1, ZNF800, GRM8BICF2P594418 15 58424953 4.48E−04 2 A 0.19 0.10 2.07 No LD FAM198B,TMEM144, RXFP1, ETFDH, PPID, FNIP2 BICF2G630422966 15 58852255 4.41E−043 A 0.41 0.28 1.80 58852255-58978372 RAPGEF2, BICF2G630422956 1558891376 4.41E−04 3 A 0.41 0.28 1.80 C15H4orf45 BICF2G630422900 1558967776 2.04E−04 3 G 0.37 0.24 1.87 BICF2G630422895 15 589783721.69E−04 3 A 0.37 0.24 1.89 BICF2P880005 17 20749191 2.22E−04 1, 2 G0.44 0.31 1.75 No LD KLHL29 BICF2P1121006 18 54279578 1.11E−04 1, 2, 3 A0.63 0.42 2.28 No LD CCDC87, DPP3, NPAS4, SLC29A2, BRMS1, RIN1, CD248,CNIH2, RAB1B, KLC2, PACS1, GAL3ST3, SART1, TSGA10IP, C18H11orf68, FOSL1,CTSW, CFL1, SNX32, OVOL1, RNASEH2C, KAT5, RELA, SIPA1, PCNXL3, MAP3K11,KCNK7, EHBP1L1, LTBP3, SCYL1 BICF2P888055 20 13815084 3.25E−04 3 A 0.730.62 1.71 No LD None BICF2P582174 20 14124824 3.76E−04 3 C 0.82 0.731.61 14118014-14124824 None TIGRP2P270462 20 15036973 8.51E−05 3 G 0.850.75 1.88 14838270-15053718 EDEM1, ARL8B BICF2P716829 20 150481919.85E−05 3 G 0.85 0.75 1.86 BICF2P1462185 20 15053718 4.90E−05 3 A 0.850.74 1.90 BICF2P178583 20 30190042 1.41E−04 1, 2 G 0.48 0.31 2.0930039696-30190042 ADAMTS9, PRICKLE2, PSMD6, ATXN7, THOC7, SNTN, SYNPRBICF2S2328420 20 51150968 2.77E−04 3 G 0.75 0.60 1.96 No LD OR12C09,OR4B10, ADGRE2, ZNF333, EMR3, CLEC17A, NDUFB7, TECR, DNAJB1, GIPC1,PTGER1, PKN1, DDX39A, CD97, LPHN1, ASF1B, PRKACA, SAMD1, PALM3, IL27RA,RLN3, RFX1, DCAF15, PODNL1, CC2D1A, C20H19orf57, NANOS3, ZSWIM4,C20H19orf53 BICF2P420488 20 52326317 3.84E−04 3 G 0.85 0.75 1.8451873051-52326317 MRI1, CCDC130, CACNA1A, NACC1, NFIX, DAND5, RAD23A,CALR, SYCE2, KLF1, MAST1, RNASEH2A, PRDX2, HOOK2, BEST2, TNPO2, WDR83,MAN2B1, ZNF791, ZNF709, ACP5, ELOF1 TIGRP2P277002 20 55563965 2.25E−041, 2 A 0.25 0.10 2.98 No LD INSR, ARHGEF18, PEX11G, C20H19orf45, MCOLN1,PNPLA6, CAMSAP3, XAB2, PCP2, STXBP2, RETN, C20H19orf59, FCER2, CLEC4G,CD209, EVI5L, LRRC8E, MAP2K7, SNAPC2, CTXN1, TIMM44, ELAVL1, FBN3,CERS4, CD320, RAB11B, MARCH2, PRAM1, ZNF414, MYO1F BICF2P111342 217150110 1.25E−04 1, 2 A 0.31 0.18 2.04 No LD None BICF2G630658881 217582214 1.09E−04 1, 2, 3 G 0.49 0.32 2.12 7582214-8382709 JRKL, CCDC82,BICF2G630658668 21 8205285 4.03E−04 3 G 0.49 0.32 2.00 MAML2, MTMR2,BICF2G630658620 21 8382709 2.90E−04 3 G 0.50 0.33 2.02 CEP57, FAM76B,SESN3 BICF2G630658768 21 8033283 4.09E−04 1, 2 C 0.27 0.14 2.288033283-8061623 JRKL, CCDC82, BICF2G630658756 21 8040746 4.73E−04 1 A0.26 0.13 2.30 MAML2, MTMR2, BICF2G630658723 21 8061623 4.09E−04 1, 2 G0.27 0.14 2.28 CEP57, FAM76B, SESN3 BICF2S2442023 21 43533585 3.19E−04 3G 0.50 0.34 1.93 43507320-43533585 NUCB2, NCR3LG1, KCNJ11, ABCC8, USH1C,OTOG, MYOD1, KCNC1, SERGEF, TPH1, SAAL1, SAA1, HPS5, GTF2H1, LDHABICF2S2361376 21 43752575 1.76E−04 1, 2, 3 A 0.60 0.42 1.9743752575-43808389 LDHC, TSG101, UEVLD, SPTY2D1, TMEM86A, IGSF22, PTPN5BICF2P321064 21 44627903 1.66E−04 1, 2, 3 G 0.38 0.24 1.99 No LDMRGPRX2, ZDHHC13, CSRP3, E2F8, NAV2 TIGRP2P293361 22 42354230 2.27E−04 2A 0.49 0.36 1.74 No LD SLITRK5 TIGRP2P297337 22 58201452 1.08E−04 1, 2,3 A 0.44 0.27 2.20 No LD EFNB2, ARGLU1 BICF2G630375268 23 333763833.48E−04 3 A 0.74 0.63 1.75 No LD TEMEM108, BFSP2, CDV3, TOPBP1, TF,SRPRB, RAB6B, C23H3orf36, SLCO2A1 BICF2S23730962 23 53809871 2.93E−04 2A 0.87 0.78 1.88 No LD TIPARP, LEKR1, CCNL1, VEPH1, PTX3 BICF2G63050222524 23992936 4.86E−04 2 G 0.92 0.81 2.84 30241088-30245795 BPI LBP,BICF2G630500368 24 30241088 2.76E−07 1, 2, 3 G 0.83 0.66 2.56 RALGAPB,ADIG, BICF2G630500363 24 30245795 1.82E−06 1, 2, 3 G 0.80 0.62 2.44ARHGAP40, SLC32A1, ACTR5, PPP1R16B, FAM83D, DHX35 BICF2G630500835 2429648925 1.29E−04 2, 3 A 0.79 0.67 1.90 No LD CTNNBL1, VSTM2L, TTI1,RPRD1B, TGM2, KIAA1755 BICF2P544126 24 29772193 4.09E−05 3 G 0.94 0.872.28 29772193-29794411 BPI LBP, BICF2S24111418 24 29794411 4.09E−05 3 A0.94 0.87 2.28 RALGAPB, ADIG, ARHGAP40, SLC32A1, ACTR5, PPP1R16B,FAM83D, DHX35, CTNNBL1, VSTM2L, TTI1, RPRD1B, TGM2, KIAA1755BICF2G630799191 26 22848912 1.33E−04 3 G 0.61 0.45 1.92 No LD ADRBK2,MYO18B, SEZ6L, ASPHD2, HPS4, SRRD, TFIP11, TPST2, CRYBB1, CRYBA4BICF2P792911 26 22894961 8.55E−05 1, 2, 3 G 0.44 0.27 2.14 No LD ADRBK2,MYO18B, SEZ6L, ASPHD2, HPS4, SRRD, TFIP11, TPST2, CRYBB1, CRYBA4BICF2S2356299 27 30557856 2.21E−05 2, 3 A 0.43 0.27 2.03 No LD AEBP2,PLEKHA5 BICF2P1332722 27 30603252 2.19E−04 1, 2 G 0.78 0.60 2.33 No LDAEBP2, PLEKHA5 BICF2P1047447 27 31108106 2.80E−04 3 A 0.52 0.38 1.75 NoLD CAPZA3, PLCZ1, PIK3C2G BICF2P487060 27 33778510 4.55E−04 3 C 0.400.27 1.81 33778510-33809600 MGST1, SLC15A5, DERA, STRAP, EPS8, PTPROBICF2P599881 27 35600038 2.66E−04 3 G 0.85 0.74 1.97 No LD PLBD1,ATF7IP, GRIN2B BICF2P1410038 27 37697040 3.33E−04 3 C 0.70 0.56 1.82 NoLD BCL2L14, ETV6, TAS2R42, CAFA- T2R67, CAFA- T2R43, CAFA- T2R12,TAS2R10, TAS2R9, TAS2R8, TAS2R7, CSDA, STYK1, MAGOHB, KLRA1BICF2S23535135 27 37814333 1.13E−04 3 A 0.31 0.20 1.83 No LD BCL2L14,ETV6, TAS2R42, CAFA- T2R67, CAFA- T2R43, CAFA- T2R12, TAS2R10, TAS2R9,TAS2R8, TAS2R7, CSDA, STYK1, MAGOHB, KLRA1 TIGRP2P355298 27 391342911.31E−04 3 G 0.74 0.57 2.16 No LD KLRK1, KLRD1, GABARAPL1, TMEM52B,OLR1, CLEC7A, CLEC1A, CLEC9A, CLEC12A, KLRF2, CD69, CLEC2D, KLRB1, PZPBICF2S23255928 27 39211186 1.10E−04 2, 3 A 0.23 0.13 2.0739211186-39217437 A2M BICF2P526639 27 39217437 4.12E−05 2, 3 G 0.23 0.122.18 BICF2S23152419 27 39428263 1.77E−05 3 A 0.79 0.64 2.1339428263-39445306 KLRG1, M6PR, BICF2P491441 27 39434491 3.34E−04 3 G0.78 0.63 2.06 PHC1, A2ML1 TIGRP2P355396 27 39445306 3.34E−04 3 A 0.780.63 2.06 BICF2P337576 27 39463031 2.35E−04 3 G 0.70 0.55 1.9239346073-39511019 RMKLB BICF2P794117 27 39511019 3.76E−04 3 C 0.71 0.562.00 BICF2P155064 27 39526004 4.68E−04 3 G 0.21 0.14 4.78 No LD NoneBICF2P992747 27 39580957 1.65E−04 3 A 0.77 0.62 2.07 No LD MFAP5BICF2S23652189 27 39644847 3.99E−04 3 G 0.31 0.21 1.64 39606871-39644847AICDA, APOBEC1, DPPA3 TIGRP2P362234 28 41376035 3.71E−04 2 G 0.87 0.742.37 41376035-41377128 MGMT, EBF, BICF2S23346408 28 41377128 1.25E−04 1,2 A 0.87 0.73 2.53 GLRX3 BICF2S23713161 29 20562935 4.59E−04 2 G 0.750.63 1.74 No LD CPA6, PREX2, C29H8orf34 BICF2S23410873 29 206728642.18E−04 2, 3 C 0.82 0.66 2.35 20672864-20703202 CPA6, PREX2,BICF2P1135545 29 20703202 2.81E−04 2, 3 A 0.82 0.67 2.31 C29H8orf34BICF2P483191 29 21601273 2.31E−05 1, 2, 3 C 0.73 0.51 2.54 No LD SULF1,SLCO5A1, PRDM14, NCOA2, TRAM1 BICF2P139173 29 22067666 3.59E−04 2 A 0.500.36 1.79 22050835-22191229 SULF1, SLCO5A1, BICF2P361907 29 221912293.47E−04 2 G 0.50 0.36 1.75 PRDM14, NCOA2, TRAM1 BICF2P456086 2923130206 4.85E−04 3 A 0.88 0.71 3.04 No LD XKR9, EYA1 BICF2P662502 2926040013 3.24E−04 1, 2 G 0.90 0.73 2.87 No LD JPH1, GDAP1, PI15,CRISPLD1 BICF2G630412697 30 3126573 7.22E−05 1, 3 G 0.96 0.86 4.23 No LDCOR4F22P, COR4F25, COR4F24P, COR4T2P BICF2S2356993 31 12920807 2.25E−042, 3 A 0.25 0.14 2.04 No LD None BICF2P287265 31 30555902 4.38E−04 3 G0.96 0.87 3.38 No LD MIS18A, MRAP, URB1, EVA1C, C31H21orf59, SYNJ1,PAXBP1, C31H21orf62, OLIG2, OLIG1, DONSON, ATP50, IFNAR2, IL10RBBICF2S23054250 35 26868731 2.20E−04 2, 3 A 0.49 0.33 1.97 No LD LRRC16A,SCGN, HIST1H2AA, HIST1H2BA, SLC17A4, SLC17A1, TRIM38, HFE, HIST1H1T,BTN2A2, BTN1A1 BICF2P1086740 37 26916351 4.34E−04 2 G 0.41 0.24 2.18 NoLD SMARCAL1, RPL37A, IGFBP2, IGFBP5, TNP1 BICF2P708698 37 269244731.53E−04 2 A 0.63 0.45 2.06 No LD SMARCAL1, RPL37A, IGFBP2, IGFBP5, TNP1Note: OR odds ratio calculated from PLINK. LMM Linear mixed model1—GCTA, 2—GEMMA, 3—PUMA. SNP position and genomic regions are based onCanFam 2.0.

TABLE 3 Statistical Power and odds ratio correction for anteriorcruciate ligament rupture GWAS risk loci identified by GEMMA in theLabrador Retriever INPower Estimated Corrected Corrected number of SNPCFA MAF Beta Significance OR f(u) Power OR Power loci BICF2S23638642 10.116 0.2327823 4.95E−04 3.07 0.07 0.986 1.38 0.188 6.1 BICF2S22959529 10.07595 0.3139731 1.58E−04 4.78 0.03 0.991 1.83 0.304 3.9 BICF2P247448 20.3957 −0.1791686 1.48E−04 1.67 0.55 0.835 1.23 0.224 2.6BICF2G630464857 2 0.08439 0.3041543 2.00E−04 3.69 0.04 0.976 1.58 0.2293.7 BICF2P564273 3 0.4072 −0.173683 1.07E−04 2.16 0.52 0.993 1.47 0.6113.5 BICF2G630349775 3 0.1181 0.2355141 4.26E−04 2.9 0.07 0.975 1.370.181 5.5 BICF2S24415473 3 0.3165 0.1940324 7.07E−05 1.97 0.26 0.963 1.50.594 2.0 BICF2G630359517 3 0.4198 0.1503946 3.77E−04 1.82 0.36 0.9371.2 0.182 6.2 BICF2G63058646 4 0.07806 0.3008072 1.76E−04 3.71 0.040.977 1.62 0.251 4.1 BICF2P295392 4 0.06962 0.2949176 4.61E−04 3.07 0.040.908 1.39 0.138 5.9 BICF2G630175389 4 0.2616 −0.2019631 5.87E−05 2.280.68 0.984 1.69 0.773 2.3 BICF2P498515 6 0.09958 0.2861419 7.89E−05 3.110.06 0.978 1.93 0.573 3.4 BICF2P1072682 7 0.1957 0.2030288 4.09E−04 2.420.14 0.986 1.3 0.208 4.1 BICF2P1208798 9 0.4388 0.2050435 5.49E−05 2.270.36 0.998 1.7 0.87 1.9 BICF2P890246 9 0.2764 −0.2119253 3.23E−05 2.990.36 1 2.19 0.996 2.5 BICF2P65003 12 0.3481 −0.2006769 3.15E−04 1.610.61 0.75 1.16 0.134 2.5 BICF2G630606359 13 0.3825 −0.1598137 4.61E−041.67 0.57 0.827 1.16 0.137 4.8 BICF2P594418 15 0.1414 0.2242702 4.48E−042.07 0.1 0.84 1.24 0.129 4.6 BICF2P880005 17 0.3671 0.1722047 2.22E−041.75 0.31 0.89 1.21 0.186 4.0 BICF2P1121006 18 0.4916 −0.209966 1.11E−042.28 0.42 0.998 1.5 0.663 1.6 BICF2P178583 20 0.3776 0.176725 1.41E−042.09 0.31 0.989 1.36 0.408 3.4 TIGRP2P277002 20 0.1624 0.23452412.25E−04 2.98 0.1 0.996 1.45 0.301 3.0 BICF2P111342 21 0.05907 0.20313081.25E−04 2.04 0.18 0.946 1.38 0.34 3.1 BICF2G630658881 21 0.39030.1872034 1.09E−04 2.12 0.32 0.991 1.45 0.559 2.0 BICF2G630658768 210.1899 0.2129482 4.09E−04 2.28 0.14 0.97 1.28 0.189 3.9 BICF2S2361376 210.4873 0.1678988 1.76E−04 1.97 0.42 0.979 1.28 0.304 3.8 BICF2P321064 210.2975 0.1845238 1.66E−04 1.99 0.24 0.962 1.29 0.266 3.7 TIGRP2P29336122 0.4156 0.1686531 2.27E−04 1.74 0.36 0.897 1.21 0.195 4.0TIGRP2P297337 22 0.3397 0.1873248 1.08E−04 2.2 0.27 0.993 1.48 0.573 2.3BICF2S23730962 23 0.1857 −0.2162607 2.93E−04 1.88 0.78 0.789 1.22 0.1523.8 BICF2G630502225 24 0.1435 −0.223976 4.86E−04 2.84 0.81 0.978 1.350.254 4.4 BICF2G630500835 24 0.2827 −0.1744313 1.29E−04 1.9 0.67 0.9121.33 0.332 4.4 BICF2G630500368 24 0.27 −0.2641268 2.76E−07 2.56 0.660.997 2.44 0.995 0.8 BICF2P792911 26 0.3432 0.1878882 8.55E−05 2.14 0.270.989 1.53 0.645 2.2 BICF2S2356299 27 0.339 0.1673825 2.21E−05 2.03 0.270.977 1.71 0.842 4.2 BICF2P1332722 27 0.3228 −0.1794126 2.19E−04 2.330.6 0.996 1.34 0.378 3.8 BICF2P526639 27 0.1695 0.2271469 4.12E−05 2.180.12 0.927 1.71 0.626 3.5 BICF2S23346408 28 0.211 −0.2180301 1.25E−042.53 0.73 0.989 1.53 0.55 2.2 BICF2S23713161 29 0.3186 −0.15927044.59E−04 1.74 0.63 0.85 1.18 0.152 6.2 BICF2S23410873 29 0.2722 −0.175162.18E−04 2.35 0.66 0.992 1.34 0.351 4.6 BICF2P483191 29 0.3948−0.2013067 2.31E−05 2.54 0.51 1 2.02 0.982 2.3 BICF2P361907 29 0.41980.1766305 3.47E−04 1.75 0.36 0.903 1.19 0.17 2.8 BICF2P662502 29 0.2025−0.1924562 3.24E−04 2.87 0.73 0.997 1.39 0.372 4.9 BICF2S2356993 310.1857 0.2003128 2.25E−04 2.04 0.14 0.907 1.27 0.18 4.7 BICF2S2305425035 0.3945 0.1628769 2.20E−04 1.97 0.33 0.975 1.26 0.258 4.1BICF2P1086740 37 0.3143 0.1743002 4.34E−04 2.18 0.24 0.989 1.26 0.2274.1 BICF2P708698 37 0.4768 −0.1611546 1.53E−04 2.06 0.45 0.989 1.330.389 4.1 Note: OR odds ratio calculated from PLINK. Corrected OR wascalculated using an approximate conditional likelihood approach [GhoshA, Zou F, Wright F A. Estimating odds ratios in genome scans: Anapproximate conditional likelihood approach. Am J Human Genet. 2008; 82:1064-1074]. GWAS data were derived using GEMMA. For each risk locusdetected by GEMMA, the total number of risk loci was estimated usingINPower [Park J-HM, Wacholder S, Gail M, Peters U, Jacobs K B, Chanock SJ, et al. Estimation of effect size distribution from genome-wideassociation studies and implications for future discoveries. Nat Genet.2010; 42: 570-575].

With the Labrador Retriever breed, associated regions (P<5.0E-04)explained the approximately half of the phenotypic variance in the ACLrupture trait (FIG. 3). For GCTA, 36 loci in 72.7 Mb of the genomeexplained 48.09% of the phenotypic variance. For GEMMA, 47 loci in 82.7Mb of the genome explained 55.88% of the phenotypic variance. For PUMA,65 loci in 86.58 Mb of the genome explained 50.28% of the phenotypicvariance in the ACL rupture trait.

We identified 129 SNPs associated with canine ACL rupture. By using LDclumping, we found that these SNPs reside in 98 loci. Five of theseregions were located in uncharacterized or non-coding regions of thegenome. A SNP on CFA24 met genome-wide significance for LMM associationanalysis with GEMMA and PUMA, but not GCTA (P=3.63E-06). This SNPresides in a 5 kB haplotype block with two other SNPs. Ten genes arelocated within the locus defined by 500 kB flanking regions includingbactericidal/permeability-increased protein (BPI), lipopolysaccharidebinding protein (LBP), Ral GTPase activation protein beta subunit(RALGAPB), adipogenin (ADIG), rho GTPase activating protein 40(ARHGAP40), solute carrier family 32, member 1 (SLC32A1), ARPSactin-related protein 5 (ACTR5), protein phosphatase 1, regulatorysubunit 16B (PPP1R16B), family with sequence similarity 83, member D(FAM83D), and DEAH (Asp-Glu-Ala-His; SEQ. ID. NO: 1) box polypeptide 35(DHX35). Although many risk loci contained large numbers of genes, fiveloci did not (Table 1, Table 2), suggesting these SNPs may have aregulatory function on gene expression (rSNPs).

Power analysis of our GWAS data set using INPower estimates that 172loci explain the genetic contribution to ACL rupture in the LabradorRetriever. See Table 3.

Risk loci clearly distinguish ACL rupture cases from controls:

To evaluate the cumulative effects of associated ACL rupture risk loci,we used a genetic risk scoring approach using a simple allele count(cGRS) or a weighted approach (wGRS). We found significant differencesin the number of risk alleles in cases and controls for GCTA(P<2.2E-16), GEMMA (P<2.2E-16), and PUMA (P<2.2E-16) (Table 4), with ashift to increased numbers of risk alleles in the cases. See FIGS. 4 and5. When the odds ratios according to the wGRS quartiles for each LMMwere calculated, there was a significant increase in ACL rupture oddsratios with increasing wGRS quartile for all three LMM, using the firstwGRS quartile as a reference (FIGS. 4 and 5)

TABLE 4 Genetic risk scoring in anterior cruciate ligament rupture caseand control Labrador Retriever dogs using GWAS associated SNPs fromlinear mixed model analysis Number of Risk alleles LMM Control CaseSignificance GCTA 24 (16, 40) 35 (24, 43) P < 2.2E−16 GEMMA 33 (22, 43)45 (29, 55) P < 2.2E−16 PUMA 62 (37, 84) 77 (56, 99) P < 2.2E−16 Note:Data represent median (range) for allele counting (cGRS). LMM Linearmixed models used were GCTA, GEMMA, PUMA. The Mann-Whitney U test wasused to determine significance.

AUC differences between cGRS and wGRS were small and we found that therewere no significant differences in ROC AUC for cGRS and wGRS for any ofthe three LMM analyses. For both cGRS and wGRS analyses, GCTA and GEMMAyielded increased ROC AUC values, when compared with PUMA. Overall, cGRSfor GEMMA yielded the highest AUC at 0.9634 (Table 5).

TABLE 5 Receiver operating characteristic (ROC) analysis of genetic riskscoring in anterior cruciate ligament rupture case and control LabradorRetriever dogs using GWAS associated SNPs from linear mixed modelanalysis Area under the ROC curve Significance 95% confidence 95%confidence LMM cGRS interval wGRS interval GCTA 0.9487 0.9191-0.97250.9464 0.9183-0.9694 GEMMA 0.9634 0.9369-0.9824 0.9601  0.933-0.9801PUMA 0.8842* 0.8356-0.9158 0.8909* 0.8458-0.9263 Note: LMM Linear mixedmodels used were GCTA, GEMMA, PUMA. *Significantly different from GCTAand GEMMA (P < 0.005 for cGRS and P < 0.05 for wGRS).GWAS pathways are enriched for aggrecan signaling:

Functional annotation clustering using DAVID revealed association with acluster of four genes (CD209, ACAN, KLRA1, KLRD1) (P<2.3E-03,P_(corr)=0.059) that includes aggrecan (ACAN), a large structuralprotein that stabilizes the collagen network in ligament matrix [30].Using INRICH, we identified enrichment for a single set of genes (TTR,SLC9A5, SLC10A1, SLC37A4, SLC6A1, AQP9. GABRP, GJB1, KCNJ3, ALB, GABRB3,P2RX1, SLC16A2) (P<4.0E-4, P_(corr)=0.07). This pathway primarilyconsists of genes encoding membrane transport proteins with a wide rangeof physiological functions including pH regulation, glucose homeostasis,signal transduction.

ACL rupture in the Labrador Retriever has moderate heritability:

Using a Bayesian method, narrow sense genetic heritability of ACLrupture was estimated at 0.538. Broad sense heritability from pedigreeswas estimated at 0.521. After correction to the liability scale for abinary trait, these estimates were 0.493 and 0.476, respectively.

Discussion:

By undertaking a within-breed GWAS in the Labrador Retriever, we found98 regions of association with the trait, suggesting that ACL rupture isa complex, potentially highly polygenic condition. These loci explainedbetween 48% and 56% of the disease risk phenotype, depending on whichLMM was used for the association analysis, suggesting that inheritedfactors make an important contribution to the disease. We estimatednarrow sense genetic heritability to be 0.49 and broad senseheritability to be 0.48, higher values than past estimates in theNewfoundland and Boxer breeds. Wilke V L, Conzemius M G, Kinghorn B P,Macrossan P E, Cai W, Rothschild M F. Inheritance of rupture of cranialcruciate ligament in Newfoundlands. J Am Vet Med Assoc. 2006; 228:61-64. Nielen A L, Janss L L, Knol B W. Heritability estimations fordiseases, coat color, body weight, and height in a birth cohort ofBoxers. Am J Vet Res. 2001; 62: 1198-1206.

Our study population of Labrador Retriever dogs was typical of thegeneral population, with an approximately equal numbers of male andfemale dogs and a large majority of the dogs being neutered bycastration or ovariohysterectomy, respectively. ACL rupture in dogs isan acquired condition. In the present study, ACL rupture cases weremiddle-aged dogs typically, with a mean age of 6.0 years. In dogs, lossof sex steroids through neutering is a risk factor for ACL rupture[Whitehair J G, Vasseur P B, Willits N H. Epidemiology of cranialcruciate ligament rupture in dogs. J Am Vet Med Assoc. 1993; 203:1016-1019]. In human beings, ACL rupture is predisposed to femaleathletes [Sutton K M, Bullock J M. Anterior cruciate ligament rupture:Differences between males and females. J Am Acad Orthop Surg. 2013; 21:41-50]. Knee laxity in women is lowest in the follicular phase of themenstrual cycle (low estrogen), when ACL rupture is most common. BeynnonB D, Johnson R J, Braun S, Sargent M, Bernstein I M, et al. Therelationship between menstrual cycle phase and anterior cruciateligament injury. Am J Sports Med. 2006; 34: 757-764. Hewett T E, ZazulakB T, Myer G D. Effects of the menstrual cycle on anterior cruciateligament injury risk. Am J Sports Med. 2007; 35: 659-668. [33,34]. Thissuggests that the influence of sex steroid levels on ACL laxity in bothspecies may influence accumulation of matrix damage over time andconsequently risk of rupture.

Because of the high LD within breeds of dogs, risk loci often containedlarge numbers of genes. However, several risk loci appeared to containrSNPs located in gene deserts in intergenic regions of the genomeof >500 kb that lack annotated genes or protein-coding sequences.Schierding W, Cutfield W S, O'Sullivan J M. The missing story behindgenome wide association studies: single nucleotide polymorphisms in genedeserts have a story to tell. Front Genet. 2014; 5: 39. Complex traitdisease is caused by disturbance to biological networks, not isolatedgenes or proteins. Regulatory SNPs can influence gene expression througha number of mechanisms that include the three dimensional organizationof the genome, RNA splicing, transcription factor binding, DNAmethylation, and long non-coding RNAs (lncRNA). Huang Q. Genetic studyof complex diseases in the post-GWAS era. J Genet Genomics. 2015; 42:87-98. Investigation of SNPs associated with complex trait disease indogs with potential regulatory function through expressed quantitativetrait loci (eQTL) studies or other methods is currently lacking.

One locus consisting of a 5 kb haplotype block with two other SNPs onCFA 24 met genome-wide significance in the present study. Ten genes wereidentified in this block with diverse physiological effects on cellularand tissue homeostasis. For example, ACTR5 plays an important role inchromatin remodeling during transcription, DNA repair, and DNAregulation. DHX35 encodes an ATP-ase that plays a role in RNA splicing[40] and RALBAPB as well as FAM83D are both important for mitoticregulation. While a relationship between cellularhomeostasis/proliferation and ACL rupture has not been established, itis feasible that aberrations in the genes that govern these processescould have a wide range of effects that may alter ligament tissueintegrity. Other genes in this block include LBP and BPI, which have inimportant function regarding immuno-stimulatory capacity of innateimmune mechanisms. Certain LBP genotypes have been associated withchronic inflammatory disease [Schumann R. Old and new findings onlipopolysaccharide-binding protein: a soluble pattern-recognitionmolecule. Biochem Soc Trans. 2011:39: 989-993]. Notably, PPP1R16Bencodes a protein that promotes angiogenesis through inhibition ofPhosphatase and tensin homolog (PTEN) [Obeidat M, Li L, Ballermann B.TIMAP promotes angiogenesis by suppressing PTEN-mediated Akt inhibitionin human glomerular endothelial cells. Am J Physiol Renal Physiol.2014:307: F623-F633]. The angiogenesis-associated signaling cascade isimportant for ligament matrix remodeling following mechanical loading,and variations in this cascade have been associated with non-contact ACLrupture risk [Rahim M, Gibbon A, Hobbs H, vander Merwe W, Posthumus M,Collins M, et al. The association of genes involved in theangiogenesis-associated signaling pathway with risk of anterior cruciateligament rupture. J Orthop Res. 2014; 32: 1612-1618].

To further investigate the large number of genes we identified withinrisk loci, we also undertook pathway analysis of our data using twodifferent methods. Pathway analysis using DAVID revealed an associationwith a cluster of four carbohydrate-binding protein genes includingaggrecan (ACAN). Aggrecan is a large aggregating proteoglycan that,through binding to fixed charged groups, maintains osmotic pressure incollagenous tissues to promote water retention. Tissue hydration isimportant for efficient distribution of load and for the ability ofcells to accomplish repair. Equine degenerative suspensory ligamentdesmitis (DSLD), a debilitating disorder of horses that leads tocollagen disruption and eventual failure of the suspensory ligament, isassociated with a 15-fold increase in aggrecan content of affectedligaments [Plaas A, Sandy J D, Liu H, Diaz M A, Schenkman D, Magnus R P,et al. Biochemical identification and immunolocalization of aggrecan,ADAMTS5 and inter-alpha-tryspin-inhibitor in equine degenerativesuspensory ligament desmitis. J Orthop Res. 2011; 29: 900-906].Moreover, recent work has linked human ACAN rs1516797 with the risk ofACL injury in both male and female participants [Mannion S, MtintsilanaA, Posthumus M, van der Merve W, Hobbs H, Collins M, et al. Genesencoding proteoglycans are associated with risk of anterior cruciateligament ruptures. Br J Sports Med. 2014; 48: 1640-1646]. A separatestudy revealed ACAN gene expression is up-regulated in ACL samples fromfemale compared to male patients that have undergone ACL repair surgery,suggesting a possible etiology for the observed sex differences amongpatients with ACL injury. The precise mechanism by which ACANup-regulation may lead to ligament weakening is currently unclear,though a structural change appears to be the most likely etiology.

We also tested genomic regions associated with ACL rupture for gene setenrichment using INRICH. One pathway, module 415 from the MolecularSignatures Database, was inflated. This pathway included 13 genes, mostof which encode membrane transport proteins with various physiologicalroles. GJBI is a member of the large connexin family and encodesconnexin 32, a gap junction protein that has been implicated in theregulation of collagen synthesis and the matrix remodeling response tomechanical loading of tendon. Young N J, Becker D L, Fleck R A, GoodshipA E, Patterson-Kane J C. Maturational alterations in gap junctionexpression and associated collagen synthesis in response to tendonfunction. Matrix Biol 2009; 28: 311-323. Waggett A D, Benjamin M, RalphsJ R. Connexin 32 and 43 gap junctions differentially modulate tenocyteresponse to cyclic mechanical load. Eur J Cell Biol. 2006; 85:1145-1154. Other genes in this module are associated with centralnervous system function. SLC6A1, GABRP, and GABRB3 are all associatedwith GABA signaling and mutations in TTR and have been associated withsensorimotor polyneuropathy. Previous work has suggested a role forneurological pathways in susceptibility to ACL rupture in Newfoundlanddogs. Baird A E G, Carter S D, Innes J F, Ollier W, Short A. Genome-wideassociation study identifies genomic regions of association for cruciateligament rupture in Newfoundland dogs. Animal Genetics. 2014; 45:542-549.

ACL rupture GRSs were calculated for each dog to determine thecumulative effect of ACL rupture-associated loci on disease risk. Whileprevious work found that wGRS better accounted for genetic risk [Chen H,Poon A, Yeung C, Helms C, Pons J, Bowcock A M, et al. A genetic riskscore combining ten psoriasis risk loci improves disease prediction.PLoS One. 2011; 6: e19454], our study found no difference between cGRSand wGRS for any of the LMMs used. This is consistent with the idea thatthe ACL rupture phenotype is associated with a large number of geneticloci with small effects. In diseases with genetic loci with largeeffects, wGRS would more accurately represent the cumulative effect ofindividual loci on genetic risk. Overall, predictive capability of GRSis high, with a cGRS for GEMMA AUC of approximately 96%, indicating thatwe have clearly captured genetic loci that contribute to ACL rupturerisk in our LMM association analysis. Future work should includeverification of predictive capability by applying these methods to a newtest cohort of case and control dogs.

Narrow and broad sense heritability of ACL rupture was estimated at 0.49and 0.46 respectively using a Bayesian method. These estimates areconsiderably higher than restricted maximum likelihood (REML)heritability estimates that have been calculated for other breeds ofdog. It is unclear whether ACL rupture is truly more heritable in theLabrador Retriever compared to other breeds or if the higher value is areflection of the Bayesian method used. REML estimation of heritabilitywas attempted but was not successful, probably because of the size ofthe data set.

Best Linear Unbiased Prediction:

A regression analysis was performed on the n=174 dog data set usingGCTA-brand Software. The GCTA software is available online athttp://www.complextraitgenomics.com/software/gcta/. See also Yang J, LeeS H, Goddard M E and Visscher P M. GCTA: a tool for Genome-wide ComplexTrait Analysis. Am J Hum Genet. 2011 Jan 88(1): 76-82. [PubMed ID:21167468]. Specifically, a restricted maximum likelihood (REML) analysisof the genetic relationship matrix was executed, followed by a genomicbest linear unbiased prediction (gBLUP) analysis to arrive at anestimate of total genetic effect (i.e., a breeding value) for each dog.This analysis was then converted to the SNP effects. The 22 SNPs moststatistically associated with the phenotype are tabulated in Table 6.The gBLUP coefficients in Table 6 indicate that there are two SNPS thathave much larger coefficients than the rest. There are six SNPS withmuch smaller coefficients, and 14 SNPS with a coefficient ofintermediate size. (The positive or negative sign of the coefficients isnot relevant; the coefficients are ranked according to their absolutemagnitude.)

TABLE 6 BLUP Analysis Results snp chr ref allele blup BICF2S23135243 4 A0.000633434 BICF2G630371956 23 A 0.00058239 BICF2P401973 10 A−0.000398919 BICF2P526639 27 G 0.000373218 BICF2S23448539 17 A−0.000367718 BICF2G630114782 16 A −0.00036446 BICF2P890246 9 A−0.000358272 BICF2P170661 6 A 0.00034582 BICF2G630815470 16 G−0.000335841 BICF2G630815474 16 A −0.000335841 TIGRP2P78405_rs9180048 6A 0.000317864 BICF2S2356299 27 A 0.000316762 BICF2P1121006 18 G−0.000308138 BICF2P260555 27 A 0.000275776 BICF2P1465216 35 A0.000272433 BICF2P599385 35 A 0.000254262 BICF2P154295 5 C 0.00021946BICF2S23645462 18 A 0.000202448 BICF2P471347 27 A 0.000197961BICF2G630373050 23 A 0.000147584 BICF2P1126668 4 C −0.000183486BICF2P412007 6 A 2.50967E−05

Kits:

Kits are provided which contain reagents useful for determining thepresence or absence of polymorphisms appearing in the loci and/or genesrecited in Table 1. The kits are used with the methods described hereinto determine a dog's propensity to develop CCLR.

The kits typically include written instructions. The instructions mayoptionally provide calibration curves or charts for comparison with theexperimentally measured values. The kit generally includesoligonucleotide probes and/or primers that bind specifically with thecanine loci identified in Table 1 and thus function to reveal thepresence (or absence) of the corresponding SNP. An appropriate amount ofthe oligonucleotide primers is provided in one or more containers. Theprimers may also be provided in the form of a “gene chip” or addressedarray, such as (for example) those described in U.S. Pat. No. 7,510,841.In such an array, the primers or probes are immobilized on a solidsubstrate, typically in pre-determined, known locations. Theoligonucleotide primers may also be provided suspended in an aqueoussolution or as a freeze-dried or lyophilized powder. The container(s) inwhich the oligonucleotide(s) are supplied can be any conventionalcontainer that is capable of holding the supplied form, for instance,hermetically sealed pouches, microfuge tubes, ampoules, or bottles. Insome applications, pairs of primers may be provided in pre-measuredsingle use amounts in individual, typically disposable, tubes orequivalent containers. With such an arrangement, the sample to be testedfor the presence of SNPs can be added to the individual tubes andamplification carried out directly.

The amount of each oligonucleotide primer supplied in the kit can be anyappropriate amount, depending for instance on the market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each oligonucleotide primer provided wouldlikely be an amount sufficient to prime several PCR amplificationreactions. Those of ordinary skill in the art know the amount ofoligonucleotide primer that is appropriate for use in a singleamplification reaction.

In some embodiments, kits may optionally also include the reagentsnecessary to carry out nucleotide amplification reactions, including,for instance, DNA sample preparation reagents, appropriate buffers(e.g., polymerase buffer), salts (e.g., magnesium chloride), anddeoxyribonucleotides (dNTPs).

Kits may in addition include either labeled or unlabeled oligonucleotideprobes for use in detection of SNPs. In certain embodiments, theseprobes will be specific for a potential polymorphic site that may bepresent in the target amplified sequences. The appropriate sequences forsuch a probe will be any sequence that includes one or more of theidentified polymorphic sites, particularly those nucleotide positionsindicated in Table 1, such that the sequence the probe is complementaryto is a polymorphic site. As a general rule, the probes are at least 6nucleotides in length and typically shorter than roughly 50 nucleotides.The polymorphic site may occur at any position within the length of theprobe. It is often beneficial to use longer probes, in order to ensurespecificity. Thus, in some embodiments, the probe is at least 8, atleast 10, at least 12, at least 15, at least 20, or at least 30nucleotides.

It may also be advantageous to provide in the kit one or more controlsequences for use in the amplification reactions. The design ofappropriate positive control sequences is well known to one of ordinaryskill in the appropriate art. By way of example, control sequences maycomprise canine nucleic acid molecule(s) with known sequence at or nearone or more of the target SNP positions described in Table 1.

The kits may optionally include either labeled or unlabeledoligonucleotide probes for use in detection of the in vitro amplifiedtarget sequences. The appropriate sequences for such a probe will be anysequence that falls between the annealing sites of the providedoligonucleotide primers, such that the sequence to which the probe iscomplementary is amplified during the PCR reaction. In certainembodiments, these probes will be specific for a potential polymorphismthat may be present in the target amplified sequences.

It may also be advantageous to provide in the kit one or more controlsequences for use in the PCR reactions. The design of appropriatepositive control sequences is well known to one of ordinary skill in theappropriate art.

Additional components in specific kits may include instructions forcarrying out the assay described herein.

CONCLUSIONS

Ninety-eight (98) candidate loci are identified herein which areassociated in a statistically significant manner with heritablenon-contact CCLR in the Labrador retriever. Of these, the strongestsignal is located on a broad region in chromosome 24. The regionsidentified in this study are useful to guide breeding decisions.

What is claimed is:
 1. A method for breeding a dog, the methodcomprising: a) isolating genomic DNA from a first dog; b) assaying thegenomic DNA of step (a) for one or more single nucleotide polymorphisms(SNPs) selected from the group consisting of: Dog Chromosome SNPLocation BICF2G630500368 24 BICF2G630500363 24 BICF2S23152419 27BICF2S2356299 27 BICF2P483191 29 BICF2P50610 11 BICF2P890246 9BICF2S23324965 6 BICF2P544126 24 BICF2S24111418 24 BICF2P526639 27BICF2P1462185 20 BICF2P1208798 9 BICF2G630175389 4 BICF2S24415473 3BICF2G630412697 30 BICF2P498515 6 TIGRP2P270462 20 BICF2P792911 26BICF2G630810143 6 BICF2G630810159 6 BICF2P716829 20 BICF2P564273 3TIGRP2P297337 22 BICF2G630658881 21 BICF2S23255928 27 BICF2P1121006 18BICF2S23535135 27 BICF2P111342 21 and BICF2S23346408 28

c) detecting an allele that is associated with non-contact cranialcruciate ligament rupture at one or more of the SNPs recited in step (b)in the genomic DNA of step (b); and d) breeding the first dog having atleast one allele that is not associated with non-contact cranialcruciate ligament rupture at one or more of the SNPs recited in step (b)in the genomic DNA of step (b).
 2. The method of claim 1, wherein step(b) comprises assaying the genomic DNA for five or more singlenucleotide polymorphisms (SNPs).
 3. The method of claim 1, wherein step(b) comprises assaying the genomic DNA for ten or more single nucleotidepolymorphisms (SNPs).
 4. The method of claim 1, wherein step (b)comprises assaying the genomic DNA for fifteen or more single nucleotidepolymorphisms (SNPs).
 5. The method of claim 1, wherein step (b)comprises assaying the genomic DNA for twenty or more single nucleotidepolymorphisms (SNPs).
 6. The method of claim 1, wherein step (b)comprises assaying the genomic DNA for SNPs at all of: Dog ChromosomeSNP Location BICF2G630500368 24 BICF2G630500363 24 BICF2S23152419 27BICF2S2356299 27 BICF2P483191 29 BICF2P50610 11 BICF2P890246 9BICF2S23324965 6 BICF2P544126 24 BICF2S24111418 24 BICF2P526639 27BICF2P1462185 20 BICF2P1208798 9 BICF2G630175389 4 BICF2S24415473 3BICF2G630412697 30 BICF2P498515 6 TIGRP2P270462 20 BICF2P792911 26BICF2G630810143 6 BICF2G630810159 6 BICF2P716829 20 BICF2P564273 3TIGRP2P297337 22 BICF2G630658881 21 BICF2S23255928 27 BICF2P1121006 18BICF2S23535135 27 BICF2P111342 21 and BICF2S23346408 28

step (c) comprises detecting an allele that is associated withnon-contact cranial cruciate ligament rupture any of the SNPs recited instep (b) in the genomic DNA of step (b); and step (d) comprises breedingthe first dog having alleles that are not associated with non-contactcranial cruciate ligament rupture at all of the SNPs recited in step (b)in the genomic DNA of step (b).
 7. The method of claim 13, furthercomprising a) isolating genomic DNA from a second dog; b) assaying thegenomic DNA from the second dog for one or more single nucleotidepolymorphisms (SNPs) selected from the group consisting of: DogChromosome SNP Location BICF2G630500368 24 BICF2G630500363 24BICF2S23152419 27 BICF2S2356299 27 BICF2P483191 29 BICF2P50610 11BICF2P890246 9 BICF2S23324965 6 BICF2P544126 24 BICF2S24111418 24BICF2P526639 27 BICF2P1462185 20 BICF2P1208798 9 BICF2G630175389 4BICF2S24415473 3 BICF2G630412697 30 BICF2P498515 6 TIGRP2P270462 20BICF2P792911 26 BICF2G630810143 6 BICF2G630810159 6 BICF2P716829 20BICF2P564273 3 TIGRP2P297337 22 BICF2G630658881 21 BICF2S23255928 27BICF2P1121006 18 BICF2S23535135 27 BICF2P111342 21 and BICF2S23346408 28

c) detecting an allele that is associated with non-contact cranialcruciate ligament rupture at one or more of the SNPs recited in step (b)in the genomic DNA of step (b); and d) breeding the second dog with thefirst dog when the second dog has at least one allele that is notassociated with non-contact cranial cruciate ligament rupture at one ormore of the SNPs recited in step (b) in the genomic DNA of step (b). 8.The method of claim 7, wherein step (b) comprises assaying the genomicDNA of the second dog for five or more single nucleotide polymorphisms(SNPs).
 9. The method of claim 7, wherein step (b) comprises assayingthe genomic DNA of the second dog for ten or more single nucleotidepolymorphisms (SNPs).
 10. The method of claim 7, wherein step (b)comprises assaying the genomic DNA of the second dog for fifteen or moresingle nucleotide polymorphisms (SNPs).
 11. The method of claim 7,wherein step (b) comprises assaying the genomic DNA of the second dogfor twenty or more single nucleotide polymorphisms (SNPs).
 13. Themethod of claim 7, wherein step (b) comprises assaying the genomic DNAfrom the second dog for SNPs at all of: Dog Chromosome SNP LocationBICF2G630500368 24 BICF2G630500363 24 BICF2S23152419 27 BICF2S2356299 27BICF2P483191 29 BICF2P50610 11 BICF2P890246 9 BICF2S23324965 6BICF2P544126 24 BICF2S24111418 24 BICF2P526639 27 BICF2P1462185 20BICF2P1208798 9 BICF2G630175389 4 BICF2S24415473 3 BICF2G630412697 30BICF2P498515 6 TIGRP2P270462 20 BICF2P792911 26 BICF2G630810143 6BICF2G630810159 6 BICF2P716829 20 BICF2P564273 3 TIGRP2P297337 22BICF2G630658881 21 BICF2S23255928 27 BICF2P1121006 18 BICF2S23535135 27BICF2P111342 21 and BICF2S23346408 28

step (c) comprises detecting at least one allele that is associated withnon-contact cranial cruciate ligament rupture in any of the SNPs recitedin step (b) in the genomic DNA of step (b); and step (d) comprisesbreeding the first dog with the second dog when the second dog hasalleles that are not associated with non-contact cranial cruciateligament rupture at all of the SNPs recited in step (b) in the genomicDNA of step (b).